Project Abstract The focus of our study is to examine how the age suppressor protein, Klotho, is regulated with aging in the brain. When Klotho expression is eliminated, mice develop normally, but age to death by 4 months of age. This rapid deterioration is accompanied with a phenotype not unlike what is observed in aged humans (cognitive impairment, atherosclerosis, ectopic calcification, emphysema, osteoporosis, skin atrophy and hair loss, thymic involution, infertility and decreased bone mineral density). Elimination of Klotho in mice causes cognitive impairment that is associated with increased oxidative stress. In contrast, Klotho overexpressing transgenic mice live longer by up to 30% and are resistant to oxidative stress. Our group found that Klotho is downregulated in the aging non-human primate, rat and mouse brains Together, these have lead us to hypothesize that Klotho is important in brain function and its downregulation with age may be the result of oxidative stress which, if prevented, could ameliorate decline into neurodegenerative disease. The work proposed, examines regulation of the Klotho promoter and 3'UTR with age and the effect of oxidative damage to the Klotho promoter with age. We will determine whether the high GC content of the Klotho promoter makes it a target for age-related downregulation because of damage that accumulates over time because of oxidative stress. This will be done by comparing the oxidation state of the Klotho promoter to that of other genes both in vitro and in post mortem samples from aged rhesus monkey brain. We will also work to characterize the transcription factors that bind and induce activation of the Klotho promoter. The Klotho promoter does not contain the classical elements for transcription initiation. Understanding what factors are important for Klotho transcription may shed light on signaling pathways leading to Klotho activation and the role of Klotho in the normal cell. Last, we will determine whether Klotho is regulated by microRNAs (miR) and how miR change in the brain with age. MiR bind and regulate translation of mRNA and nothing is known about whether and how miR affect Klotho processing. Again, understanding the processes that regulate Klotho will enable us to have a better understanding of the processes that affect Klotho and Klotho's wider role in cellular function. The results of this work will add new knowledge on both the anti-aging gene Klotho and elucidate a possible mechanism for how oxidative damage selectively downregulates specific genes.